Project Summary/Abstract Aberrant surface glycosylation is a well-known tumor biomarker, however the functional role of the tumor glycome in regulating tumor cell phenotype remains poorly-understood. ?2-6 sialic acid (a bulky, negatively- charged sugar) is a prominent tumor-associated glycan elaborated by the ST6Gal-I glycosyltransferase. Research from our group has shown that ST6Gal-I sialylates select membrane receptors, including TNFR1, which in turn causes activation of NF-?B. ST6Gal-I is pervasively upregulated in ovarian cancer (OC), and high expression levels correlate with poor patient survival and metastasis. Furthermore, forced ST6Gal-I overexpression in multiple animal models fosters tumor initiation and progression. The central hypothesis of the current proposal is that ST6Gal-I activity contributes to OC development by promoting NF-kB-dependent signaling events that confer cancer stem cell (CSC)-like features to tumor cells. The NF-kB axis is one of the most hyperactivated pathways in CSCs, and is a well-accepted driver of the CSC phenotype. The concept that NFkB signaling and CSC behavior are regulated by tumor cell sialylation advances a new paradigm in cancer cell biology. The proposed research has two principal objectives. The first (Aim 1) is to establish that the master stem cell transcription factor, Sox2, is a key inducer of ST6Gal-I upregulation in OC. Our preliminary data show that Sox2 binds directly to the ST6Gal-I promoter to stimulate transcription. Downstream of ST6Gal-I upregulation, we postulate that ST6Gal-I-mediated receptor sialylation directs CSC reprogramming via NFkB, consequently facilitating tumor initiation. The second major goal of the proposal (Aim 2) is to define ST6Gal-I?s role in OC peritoneal dissemination, which is one of the chief contributors to patient mortality. Unlike most solid tumors, OC primarily disseminates via fluid flow throughout the peritoneal cavity rather than transit through the vasculature or lymphatics. The peritoneum has very low oxygen tension, and therefore OC cell adaptation to hypoxia is an essential element in OC progression. Recent studies from our group have shown that ST6Gal-I activity facilitates protection against hypoxia by potentiating HIF1? signaling. Aim 2 will test that premise that ST6Gal-I-mediated hypoxia adaptation is critical for OC cell survival and expansion within the peritoneal tumor microenvironment. Collectively, the proposed studies are expected to uncover a highly novel glycosylation-dependent mechanism that plays seminal roles in both early and late stages of OC development and progression.